*5.1.2* In vivo *nano-biopesticide treatment*

Nano-biopesticides can be applied to plants in the right concentration in order to protect them from seasonal diseases. These concentrations (LC 50 and LC 90) aid in the identification of specific larvae, insets, and bee attacks. Nano-biopesticides are also applied in changing environments such as temperatures, humidity, and environmental stresses. In these conditions, nano-biopesticides are directly applied

in the form of sprays to protect the plants from pest attacks. Therefore, the use of nano-biopesticides has become the most effective method in controlling the attack of animal vectors and disease-transmitting pests.

### *5.1.3 Activity against stored grain pests*

Pests of stored grains are among the most difficult to manage in an agricultural system because of their large size [94]. Recently, it has been shown that alumina, silica, SiO2, zinc, and silver nano-particles have a substantial anti-pest effect against a range of pests when combined with other chemicals [95]. According to the researchers, when sprayed on plants or crops, nano-emulsions have been shown to be efficient in deterring the attack of attack insects that cause harm to grains that have been stored for extended periods of time. The researchers discovered that nano-biopesticide emulsions effectively prevented the spread of the *Tribolium castaneum* fungus in one of the case studies they conducted [96]. In this study, oil/water emulsions of *P. anisum* essential oil (14 percent, v/v of the total coarse emulsion), ethanol (3 percent, v/v), and Tween 80 (3 percent, v/v), which together represented 20 percent (v/v) of the total coarse emulsion, as well as various components, were used. The emulsions were properly mixed and kept at 86°C for 1 hour. Separating the mixture from the water was accomplished by centrifuging it at 10,000 g for 15 minutes (which made up 80 percent of the mixture). A technique known as photon correlation spectroscopy was used to evaluate a variety of characteristics such as conductivity, zeta potential of the emulsion, and polydispersity index. Contact and digestion techniques on the insects in the medium were used to assess the insecticidal activity of the *P. anisum* emulsified with nano-particles. During the testing against the insects in the medium, the researchers discovered that the *P. anisum* emulsion incorporating nano-particles proved to be very effective. Different concentrations of nano-emulsions can be prepared by acetone in 50 mL bottles containing the 20 g mash grains. The positive and negative controls as emulsions of *P. anisum*. Different groups of 20 beetles were properly made in the sex ratio of 1:1 and then transferred to 50 mL bottles containing the treated and control grains, and each concentration was recorded as three replicates. The mortality rate was carefully recorded after treatments with 3-day intervals till 12 days, while on the other hand, the mortality rate with essential oil containing the beetles was recorded at intervals of 48, 72, and 96 hours. Those insets that were survived and were attached to grain were removed. F1 progeny were recorded 60 days after insect infestation to avoid generation overlaps, as elaborated by Tofel *et al.* [97]. Standard procedures were used to determine the LC 50 values that were used to control the attack of battles and to understand the morphological changes associated with the nano-emulsions.

## **6. Methods for nano-suspensions preparations**

Creating nanosuspensions may be accomplished using two distinct approaches, which are referred to as the bottom-up approach and top-down technology. The bottom-up approach is the more traditional way of creating nanosuspensions. In order to achieve top-down drug particle reduction, a number of techniques such as high-pressure homogenization and media milling are used. Following the bottom-up approach, pesticides (that are to be converted into nanosuspension) are solubilized in a suitable organic solvent and precipitated with the aid of a suitable stabilizer that has been dissolved in an antisolvent as a result of this solubilization and precipitation (often water). Methods such as precipitation, microemulsion, and melt emulsification, to name a few, are among the most often used in this method, and they are

described in more detail below [98, 99]. The following are some of the most important methods for the production of nanosuspensions, which are described below.
